CN105759050B - It is a kind of quantitatively to detect Troponin I content immunofluorescent reagent box and preparation method - Google Patents
It is a kind of quantitatively to detect Troponin I content immunofluorescent reagent box and preparation method Download PDFInfo
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- CN105759050B CN105759050B CN201610061086.8A CN201610061086A CN105759050B CN 105759050 B CN105759050 B CN 105759050B CN 201610061086 A CN201610061086 A CN 201610061086A CN 105759050 B CN105759050 B CN 105759050B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
Troponin I content immunofluorescent reagent box and preparation method are quantitatively detected the invention discloses a kind of, including：Troponin antibodies bulky grain fluorescent grain conjugate is prepared；The preparation of nitrocellulose filter labelled antibody；The assembling of reagent card；TNI calibration objects are prepared；Establish standard curve；Calculate the content of troponin in sample to be tested；The quantitatively detection Troponin I content immunofluorescent reagent box includes：Nitrocellulose filter, reagent and calibration object.The minimum detection limit of kit of the present invention meets the demand of clinical practice up to 0.1 ng/ml；It is needed 10 15 minutes from sample collection to testing result most fast is provided, has striven for more quality time for patient, dynamic detection can be carried out to conditions of patients.Results relevance of the present invention is good, no significant difference, and testing result accurately and reliably, can clinically be used with import substitutes, significantly reduce testing cost.
Description
Technical field
The invention belongs to technical field of biological more particularly to a kind of quantitatively detection Troponin I content immunofluorescences
Kit and preparation method.
Background technology
Troponin is troponin T（TnT）, Troponin I（TnI）And troponin C（TnC）Three kinds of subunit's groups
Into they are the main regulatory proteins of muscle, have dependence with calcium since three combines closely, muscle is controlled by the release of calcium
Contractile function.Troponin I（TnI）(cTnI) is distributed across in cardiac muscle cell, mainly the contraction of adjusting cardiac muscle, in Healthy People
The concentration of cTnI is less than 20.4pg/ml.Because specific high, there are higher sensibility, cTnI（cTnI）As the heart
One of the hypersensitivity of injury of muscle and the marker of high specific, while be also to judge myocardial damage and the classification of risks and sentence
The important biochemical indicator of disconnected prognosis.After myocardial damage, cardiac troponin complex is discharged into blood, 4 ~ 6 it is small when after, open
Beginning raises in blood, and raised cTnI can be kept for a long time in blood（6 ~ 10 days）, which provides longer
The detection phase.With wider diagnostic window：cTnI（4~10 days）, it is longest non-enzyme marker of holding time.They
In diagnostic window, the amplitude that cTnI increases is big, than 5~10 times of CK-MB high.Since in no myocardial damage, cTnI contains in blood
Amount is very low, therefore can also be used for small heart damage（MMD）Diagnosis, this is that former Enzyme target is difficult to.CTnI is also
Value with judging prognosis, to any coronary artery disease patient, even if ECG or other inspections（Such as exercise test）Feminine gender,
As long as cTnI increases, should be regarded as with high risk.It is very high that clinical research shows that cTNI detections have in acute diagnose and treat
Value, numerous section office such as surgery, internal medicine, emergency department and Experiment on therapy room can be applied.With the diagnosis index phase applied at present
Than cTNI has unique effect in diagnosis and differential diagnosis scheming necrosis.The method of detection cTNI has much at present, can not only determine
Property, it can also quantify, common method has：Gel chromatography and efficient liquid phase chromatographic analysis, enzyme linked immunosorbent assay (ELISA)
（Enzyme-Linked Immunosorbent Assay, ELISA), radiommunoassay Radioimmunoassay, RIA),
Immunoluminescence method and colloidal gold chromatography.Wherein gel chromatography and efficient liquid phase chromatographic analysis are time-consuming and are not easy to automate；ELISA
Standard measure accuracy is poor, the operating time is long, the degree of automation is low, is chiefly used in qualitative detection；Though golden mark method stability is preferable,
But sensitivity is relatively low, generally can only be qualitative, it is impossible to it is quantitative, particularly this shortcoming of poor repeatability limit its clinically should
With being particularly unsuitable for help the quantitative detection of the body fluid marker protein diagnosed to disease by accurate quantitative analysis.Exempt from
Epidemic disease chromatography is bonded respectively to the different loci of cTNI using two antigen-specific antibodies.One antibody is for fluorescence mark
Note, and another is then coated on above nitrocellulose, in labeling process, fluorescence antibody and the troponin molecule in sample
Sandwich complex is combined to form, this compound is when flowing through water-absorption fiber element film surface after combining the cTnI of sample to be tested, meeting
It is combined with coated another kind cTnI antibody on film, and is fixed on junction, after the completion of reaction, using appropriate photometer measurement
Optical signal, the compound object amount of calculations incorporated to membrane carrier.Light signal strength is directly proportional to cTNI concentration.It is established using own algorithm
Standard curve can acquire whole blood by it, unknown TNI concentration in serum or blood plasma.This method high specificity, sensibility
Expensive instrument is not required in height, is suitble to various scale medical institutions.Immunofluorescence fast detection method uses double-antibody sandwich
Immune detection and immune chromatograph testing principle can detect free Troponin I after myocardial damage.It is coated in advance on detection film
Antiantibody corresponding with the specific antibody of cTnI and the antibody kind in blood sample can be combined.After sample adds in, work as sample
Middle cTnI and the sample liquid mixing for being marked with Immunofluorescent particles, form Ag-Ab-fluorescent marker complex, this is compound
Logistics is rested on another specific antibody capture of T lines, precipitation forms macroscopic colour band through film surface." C " simultaneously
Area also always has another colour band of antiantibody formation, which is internal quality control.It is obtained through Immunofluorescence test instrument
T and C line number evidences are taken, by pre-set mathematical model, calculate corresponding detected value, the cTnI for being converted into blood sample of patient is dense
Degree, the signal depth and the concentration of cTnI in sample of colour band are proportionate.In immunoassay technology, using improved coating skill
Art, using new material with improve connected in detection reagent on carrier with sample to be checked occur immune response composition it is dense
Degree is the important channel for the sensitivity for improving clinical detection product.In immunofluorescence lateral flow technology, the fluorescent grain that uses
Size has very big related to detection sensitivity, and currently used is the particle of 150-250nm.
The method of detection cTNI is time-consuming there are gel chromatography and efficient liquid phase chromatographic analysis at present and is not easy to automate；
ELISA method dosing accuracy is poor, the operating time is long, the degree of automation is low, and sensitivity is relatively low, generally can only be qualitative, it is impossible to and it is quantitative,
Particularly this shortcoming of poor repeatability limits its application clinically, is particularly unsuitable for help by accurate quantitative analysis
Quantitative detection to the body fluid marker protein that disease is diagnosed.
The content of the invention
Troponin I content immunofluorescent reagent box and preparation side are quantitatively detected it is an object of the invention to provide a kind of
Method, it is intended to which the method for the current detection cTNI of solution is time-consuming there are gel chromatography and efficient liquid phase chromatographic analysis and is not easy to automate；
ELISA method dosing accuracy is poor, the operating time is long, the degree of automation is low, and sensitivity is relatively low, generally can only be qualitative, it is impossible to and it is quantitative,
Particularly this shortcoming of poor repeatability limits its application clinically, is particularly unsuitable for help by accurate quantitative analysis
The problem of body fluid marker protein diagnosed to disease quantitatively detects.
The present invention is achieved in that a kind of preparation method for quantitatively detecting Troponin I content immunofluorescent reagent box,
The preparation method for quantitatively detecting Troponin I content immunofluorescent reagent box comprises the following steps：
The first step, troponin antibodies-bulky grain fluorescent grain conjugate are prepared, and are using phosphate buffer density
10mM, buffer solution include 0.39 gram of sodium dihydrogen phosphate, 1.02 grams of disodium hydrogen phosphate, 0.2 gram of azoles nitrogen sodium；
It is 7.2 with salt acid for adjusting pH, adds 5 grams of BSA, make final concentration of：BSA 5mg/ml、Proclin 0. 05%
w/v；
Second step, the preparation of nitrocellulose filter labelled antibody take CN140 nitrocellulose filter 30cm, are placed in a film
It on instrument, operates and requires according to point film instrument, antibody liquid is uniformly put on film with the speed of 1ml/cm；
The nitrocellulose filter for being marked with antibody is placed in suitable cartridge back box and specifies by the 3rd step, the assembling of reagent card
Position, be sequentially placed into sample pad, then water absorption pad covers box in cartridge, be placed in humidity not higher than 20% sealing close in
It preserves；
4th step, TNI calibration objects are prepared, human troponin recombinant protein are dissolved in phosphate buffer and is added in 5% cow's serum,
The calibration object of various concentration is made；Using troponin calibration object as primary standard, using troponin kit to various concentration
Calibration object detect respectively 20 times, average is obtained, obtains the concentration of troponin calibration object：0. 2,0.67,1,3,4.8,10,
27.3 ng/ml；
5th step, establishes standard curve；Above-mentioned 6 standard items are dissolved in into the phosphate buffer containing 5% cow's serum, so
Each takes 75ul afterwards, instills in the sample well of test card, waits 10 minutes, has detector to read fluorescence signal, then brings into
Excel softwares are calculated, and obtain standard curve；
6th step is collected the data of wavelength of transmitted light by Immunofluorescence test instrument, substitutes into calibration curve y=0.9188x+
0.5444, thus calculate the content of troponin in sample to be tested.
The troponin antibodies-bulky grain fluorescent grain conjugate preparation specifically includes：
Step 1, the activation of latex particle, washing take 2% aldehyde radical base latex latex particle solution 100 μ 1, molten to latex
0.6mg/ml NaBH are added in liquid4 Solution I 1ml are placed in 37 °C of reciprocal shakers and react 2h；12000rpm is centrifuged
30min abandons supernatant, adds in the washing of 50mM pH7.2 phosphate buffers three times, precipitation is scattered in 7.2 phosphoric acid of 10mM pH and is delayed
In fliud flushing, it is 1% w/v to make latex particle concentration；
Step 2, the crosslinking of large scale latex particle troponin antibodies, with 10mM phosphate buffers by 5mg/ml sheep
Anti-human TNI polyclonal antibodies are diluted to the antibody-solutions of 1mg/ml, and 400 are added in the activation latex particle solution prepared in 1 ml
μ l antibody-solutions put 37 °C of shaking table lh, form large scale latex particle-TNI polyclonal antibodies connection compound, 12500rpm speed
Degree centrifugation 20min, abandons supernatant, and sediment is dissolved in O. 1M pH7. 4PBS buffer solutions, mixing, and 12500rpm centrifuges 20min,
Supernatant is abandoned, is then washed repeatedly twice；Final pellets are dissolved in the Proclin300's of BSA containing 4mg/ml and 0.05%
10mM phosphate buffers.
The preparation method of the latex particle comprises the following steps：
Step 1, activation, the washing of bulky grain latex particle；The amino group of large particle surface modification is activated, by breast
It is centrifuged after the activation of glue latex solution, abandons supernatant, precipitation is washed repeatedly 3 times with lavation buffer solution；Final pellets are resuspended in described
In lavation buffer solution, make the mass volume ratio final concentration of 1% of latex particle, the lavation buffer solution be selected from PBS buffer solution and
One kind in MES buffer solutions；
Step 2, Troponin I antibody coating latex particle
The latex solution after Iml activated rinses is taken, Troponin I antibody is added in, makes Troponin I antibody final concentration of 1
mg/ml；After mixing, put 437 °C of shaking tables and 218h is coated with 220rpm rotating speeds；
The Troponin I antibody not combined with latex particle is washed with PBS buffer solution, is repeated 3 times；
Closing：The latex particle of coating Troponin I antibody after washing is dissolved in comprising protectant buffer solution
In, make the final concentration of 0.1-0. 14% of mass volume ratio of the latex particle；
Closing after step 3, anti-human troponin antibodies and the combination of large scale latex particle, by large scale latex particle
With the anti-human troponin antibodies mixture, skimmed milk power 0.3% is added in, reacts 2 h at room temperature, centrifuged, removed wherein a small amount of
Sediment；
Step 4, cleaning, by the large scale latex particle formed in step 3-anti-human troponin antibodies complex solution
Supernatant is abandoned in centrifugation, adds in phosphate buffer repeated washing 3 times；Final pellets are dissolved in containing the protective agent and preservative
In buffer solution, make final concentration of 0. 04%-0.14% of mass volume ratio of the latex particle.
A kind of quantitative detection prepared such as the above-mentioned preparation method for quantitatively detecting Troponin I content immunofluorescent reagent box
Troponin I content immunofluorescent reagent box, the quantitatively detection Troponin I content immunofluorescent reagent box include：Nitric acid
Cellulose membrane, reagent and calibration object；
The reagent includes surfactant, blocking agent, preservative and the latex particle for being coated with anti-human troponin antibodies,
It is connected between the reagent moderate resistance human troponin antibody and large scale latex particle by peptide catenary system；The calibration object includes
Protective agent, preservative, buffer solution and restructuring troponin；
The mass ratio of the anti-human troponin antibodies and large scale latex particle is 1：10 - 1:100；
The final concentration of 0.04-0.4% of latex latex particle mass volume ratio of the anti-human troponin antibodies of coating.
The latex particle of a diameter of 390nm of bulky grain fluorescence carrier in the reagent；
The one kind of the reagent moderate resistance human troponin antibody in polyclonal antibody and monoclonal antibody；For mouse source,
Rabbit source or sheep source antibody；
One or more of the protective agent in bovine serum albumin(BSA), ovalbumin and gelatin, the ox blood are pure
Albumen, concentration 0.310mg/ml；
The troponin kit includes sample liquid, and sample liquid is selected from Tris-HCl buffer solutions, phosphate buffer
With the one or more in glycine buffer；Required concentration is 10mM-100mM, pH 610；The troponin sample liquid
Use phosphate buffer, concentration 10mM, pH 7.2.
The surfactant is selected from polysorbas20, tween 100；The mass volume ratio of the surfactant is 0.02-
0.4%；
One or more of the preservative in Sodium azide, thimerosal and PrOClin300；The matter of the preservative
Amount volume ratio is 0.1- 2%.
It is a kind of comprising the quantitatively detection Troponin I content immunofluorescent reagent another object of the present invention is to provide
The Immunofluorescence test instrument of quantitative detection Troponin I content immunofluorescent reagent box prepared by the preparation method of box.
It is provided by the invention quantitatively to detect Troponin I content immunofluorescent reagent box and preparation method, Troponin I
Quick in time, high sensitivity detection has very big clinical value, each advanced state of the world for rescue Patients With Myocardial Infarction life
Family, it is normal and abnormal line of demarcation all to take 0.1ng/ml substantially including China, and this requires clinical testing procedure will can
Detect the level of 0.1ng/ml, the company of countries in the world development ＆ production Troponin I detection at present is all using this standard as mesh
Mark carries out effort.Currently employed technical method is chemiluminescence, time resolution, immunofluorescence.Wherein immunofluorescence quantifies
Detection is at low cost since equipment is simple, quickly, easy to use, to become the new technology that the World Health Organization widelys popularize.But
Requirement of the kit of immunofluorescence method development ＆ production to each component is stringent, and the size of wherein fluorescent grain is exactly one
An a important parameter for determining product sensitivity.This patent can be combined using the fluorescent grain of bulky grain compared with multispecific antibody point
Son can load the high fluorescent characteristic of more fluoresceins, reach more highly sensitive detection result, can combine and more treat
It detects substance and generates more hyperfluorescence；Compared with prior art, there are following features：
1) kit of the present invention further employs large scale Immunofluorescent particles on immunofluorescence basis, significantly
Improve the detection sensitivity of reagent
,
The minimum detection limit of kit of the present invention meets the demand of clinical practice up to 0.1 ng/ml.
2) kit of the present invention can realize the flexible Quantitative detection of multisample type on immunofluorescence analysis instrument；It is commercially available
Troponin I detection is mostly detected using serum or blood plasma, it is impossible to meet clinical quick detection demand, kit of the present invention is worked as
When being used on immunofluorescence analysis instrument, adoptable sample type includes whole blood, serum or blood plasma, can be answered in clinical more section office
With.Kit of the present invention most only needs 10-15 minutes soon from sample collection to providing testing result, has striven for more treasured for patient
Your can carry out dynamic detection time to conditions of patients.
3) kit of the present invention and import reagent box are to the TNI content detection result statistical analysis of same sample, as a result
Correlation is good, no significant difference, testing result accurately and reliably,
| Concentration | It is repeated 5 times | AV | SD | C.V. | ||||
| 1.10 | 0.9300 | 1.1200 | 0.9760 | 1.1600 | 1.1050 | 1.058 | 0.099 | 9.4 |
| 5.00 | 4.7900 | 5.2900 | 5.1300 | 5.3400 | 4.7800 | 5.066 | 0.268 | 5.3 |
| 10.40 | 10.7800 | 10.1100 | 9.8900 | 10.7200 | 9.8900 | 10.278 | 0.441 | 4.3 |
It can clinically be used with import substitutes, reach C.V. at present<10% is all chemiluminescent product, chemistry
Luminous product testing is of high cost, if each chemiluminescence method testing costs of cTnI be 120 yuan, this method for 60 yuan/every time,
Testing cost can be greatly reduced.
Description of the drawings
Fig. 1 is quantitatively detection Troponin I content immunofluorescent reagent box and preparation method provided in an embodiment of the present invention
Fig. 2 is reagent principle schematic provided in an embodiment of the present invention；
It shows the internal structure that general fast diagnosis reagent closes, is set with quality control region on nitrocellulose filter, test section,
Sample pad, water absorption pad, and indicate detection liquid flow direction.
Fig. 3 is the concentration of bulky grain latex provided in an embodiment of the present invention and the linear relationship of optics OD；Show this patent
The major diameter latex that method uses linear relationship in 0-0.14% is good.
Fig. 4 is the comparison diagram of cTnI standard values provided in an embodiment of the present invention and this patent method detected value；It shows from row
The standard items for the cTnI standard items production company purchase approved in the industry are in the analysis chart measured with this patent method, statistical analysis
R=0.995 is obtained, linear relationship is good.
Fig. 5 is provided in an embodiment of the present invention and standard method (Dade) detects blood sample contrasting data figure.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The application principle of the present invention is explained in detail below in conjunction with the accompanying drawings.
Quantitative detection Troponin I content immunofluorescent reagent box one kind of the embodiment of the present invention quantitatively detects troponin
The immunofluorescent reagent box of I using bulky grain fluorescence carrier, coordinates larger aperture nitrocellulose filter, what is optimized is various
Agent prescription, blood sample need to be detected by, which making, steadily flows through film surface, and obtains highly sensitive testing result.Reagent includes surface-active
Agent, blocking agent, preservative and the latex particle for being coated with anti-human troponin antibodies, the reagent moderate resistance human troponin antibody with
It is connected between large scale latex particle particle by peptide catenary system；The calibration object includes protective agent, preservative, buffer solution and again
Group troponin.
The latex particle of a diameter of 390nm of bulky grain fluorescence carrier in the reagent.
For the nitrocellulose filter there are many NC95 of brand Germany sartorius, NC140, U.S. millipore are public
HF135, the HF180 of department, China is according to the similar film of energy company, preferred NC95.
The one kind of anti-human troponin antibodies in polyclonal antibody and monoclonal antibody in the reagent is preferably more
Clonal antibody.The troponin antibodies can be mouse source, rabbit source or sheep source antibody, preferably sheep source antibody.
The bulky grain styrene latex particle of the anti-human troponin antibodies of coating in the reagent is anti-human by preparing
Troponin antibodies mix with latex latex particle and chemically react crosslinking and be made, in anti-human troponin antibodies-glimmering
In light particle preparation, the mass ratio of the anti-human troponin antibodies and large scale latex particle is I：10 - 1:100, preferably
I:20。
The surface modification group of large scale latex particle in the reagent supplies to chemically react with anti-human troponin antibodies
There are many crosslinkings, carboxyl, amino, aldehyde radical, carbonyl, and preferred aldehyde radical, the system can improve the sensitivity of detection method, while not increase
Add non-specific interference.Antibody and particle surface are crosslinked, this mark reaction is mild, seldom inhibits antibody activity, labelled antibody is steady
It is fixed, and background level is very low, is tightly combined rapid.In addition, the combination of antibody and particle is very stable, it will not be because of the height of reaction reagent
Degree dilution and it is impacted, ensure that the accuracy of testing result.
The sensitization latex latex particle of the anti-human troponin antibodies of coating preserves formula of the liquid for optimization.
During large scale latex particle labelled antibody, activation large scale latex particle should have appropriate ratio with antibody to be marked
Example makes the large scale latex particle molecular amounts marked on each antibody molecule control in a certain range.With large scale latex
After burl closes, in the large scale latex particle combination cTNI Antibody preparations, the anti-human troponin antibodies and large scale breast
The molar ratio of glue particle preferably 1:20.
The final concentration of 0.04- of latex latex particle mass volume ratio of the anti-human troponin antibodies of coating in the reagent
0.4% preferably 0.2 % bovine serum albumin(BSA)s（BSA), ovalbumin（OVA) and gelatin, latex particle surface can be protected to be crosslinked
Antibody.One or more of the protective agent A in bovine serum albumin(BSA), ovalbumin and gelatin, preferably ox blood are pure
Albumen, preferably concentration 0.310mg/ml, 0.3-1mg/ml.
The troponin reagent includes sample liquid, wherein the buffer solution is selected from Tris-HCl buffer solutions, phosphate
One or more in buffer solution and glycine buffer, preferably phosphate buffer solution；The concentration of the sample liquid is 10mM-
100mM, preferably 10 mM, preferably pH 610,7.28. 5；The buffer solution of the Troponin I antibody mark is selected from borate
One or more in buffer solution, carbonate buffer solution, Tris-HCl buffer solutions, phosphate buffer and glycine buffer,
Preferably phosphate buffer solution；The concentration of the buffer solution is 10mM, pH 7.2.
The increased response agent is selected from polysorbas20, preferably tween 100, polysorbas20；The quality volume of the increased response agent
Than for 0.02-0.4%, preferably 0.02%.Polysorbas20 is non-ionic water-soluble polymer, there is very strong hydrophily, can be destroyed
Electron cloud and hydrated sheath around Proteins In Aqueous Solutions promote the antigen of specificity and antibody molecule close and combine to form big point
Sub- compound.
One or more of the preservative in Sodium azide, thimerosal and PrOClin300, preferably Proclin300,
The mass volume ratio of the preservative be 0.1- 2%, preferably 0. 1%.
The preparation method of the antibody and particle composites comprises the following steps：
1) activation, the washing of bulky grain latex particle, this step need the amino group by large particle surface modification to activate,
It is centrifuged after the latex latex solution of commercialization is activated, abandons supernatant, precipitation is washed repeatedly 3 times with lavation buffer solution；Last is sunk
Shallow lake is resuspended in the lavation buffer solution, makes the mass volume ratio final concentration of 1% of latex particle, the lavation buffer solution is selected from
One kind in PBS buffer solution and MES buffer solutions；
2) Troponin I antibody coating latex particle
1. taking the latex solution after Iml activated rinses, Troponin I antibody is added in, makes Troponin I antibody final concentration of
1 mg/ml；After mixing, put 437 °C of shaking tables and 218h is coated with 220rpm rotating speeds；
2. washing the Troponin I antibody not combined with latex particle with PBS buffer solution, it is repeated 3 times；
3. it closes：The latex particle of coating Troponin I antibody after washing is dissolved in comprising protectant buffering
In liquid, make the final concentration of 0.1-0. 14% of mass volume ratio of the latex particle.
3) closing after anti-human troponin antibodies and large scale latex particle combine
By the large scale latex particle and the anti-human troponin antibodies mixture, skimmed milk power 0.3%, room are added in
Temperature 2 h of lower reaction, centrifugation remove wherein a small amount of sediment；
4) clean, the large scale latex particle-anti-human troponin antibodies complex solution formed in step 3) centrifuged,
Supernatant is abandoned, adds in phosphate buffer repeated washing 3 times；Final pellets are dissolved in the buffer solution containing the protective agent and preservative
In, make final concentration of 0. 04%-0.14% of mass volume ratio of the latex particle.
The troponin immunofluorescent reagent box can quantitatively detect blood of human body on Immunofluorescence test instrument
The content of middle troponin, wherein the blood includes whole blood, serum and blood plasma.
As shown in Figure 1, the preparation side of the immunofluorescent reagent box of the quantitative detection Troponin I content of the embodiment of the present invention
Method comprises the following steps：
S101：Troponin antibodies-bulky grain fluorescent grain conjugate is prepared, and the use of phosphate buffer density is 10mM,
It is 7.2 with salt acid for adjusting pH, adds BSA and preservative, make final concentration of：BSA 5mg/ml、Proclin O. 05% (w/
v)；
S102：The preparation of nitrocellulose filter labelled antibody；
S103：The nitrocellulose filter for being marked with antibody is placed in what is specified in suitable cartridge back box by the assembling of reagent card
Position is being sequentially placed into sample pad, then water absorption pad covers box in cartridge, is placed in sealing of the humidity not higher than 20% and closes interior protect
It deposits.
The application principle of the present invention is further described with reference to specific embodiment.
First, the preparation of troponin assay kit
1st, troponin antibodies-bulky grain fluorescent grain conjugate is prepared, and the use of phosphate buffer density is 10mM, is used salt
Acid for adjusting pH is 7.2, adds BSA and preservative, is made final concentration of：BSA 5mg/ml、Proclin O. 05% (w/v).
1）Activation, the washing of latex particle take 2% aldehyde radical base latex latex particle solution（ThermolFisher,
Inc., grain size 390nm) 100 μ 1,0.6mg/ml NaBH are added in into latex solution4 Solution I 1ml are placed in 37 °C back and forth
2h is reacted in formula shaking table.12000rpm centrifuges 30min, abandons supernatant, adds in the washing of 50mM pH7.2 phosphate buffers three times, will be heavy
Shallow lake is scattered in 7.2 phosphate buffers of 10mM pH, and it is 1% (w/v) to make latex particle concentration.
2) crosslinking of large scale latex particle troponin antibodies, with 10mM phosphate buffers by 5mg/ml goat-anti people
TNI polyclonal antibodies (Biospacific companies) are diluted to the antibody-solutions of 1mg/ml, in activation glue prepared by 1 ml steps 2)
400 μ I antibody-solutions are added in newborn particle solution, put 37 °C of shaking table lh, form large scale latex particle-TNI polyclonal antibodies
Connect compound.12500rpm speed centrifuges 20min, abandons supernatant, sediment is dissolved in O. IM pH7. 4PBS buffer solutions, mixes
Even, 12500rpm centrifugation 20min abandon supernatant, then wash repeatedly twice；By final pellets be dissolved in BSA containing 4mg/ml and
The 10mM phosphate buffers of 0.05% Proclin300.
2nd, the preparation of nitrocellulose filter labelled antibody
The nitrocellulose filter for being marked with antibody is placed in the position specified in suitable cartridge back box by the 3rd, assembling of reagent card
It puts, is being sequentially placed into sample pad, then water absorption pad covers box in cartridge, be placed in sealing of the humidity not higher than 20% and close interior protect
It deposits.
4th, TNI calibration objects are prepared, and commercially available human troponin recombinant protein is dissolved in the solution of similar human serum matrix（Phosphorus
Acid buffer adds 5% cow's serum) in, the calibration object of various concentration is made.Using Roche Holding Ag's troponin calibration object of import as original
Beginning standard detects the calibration object of various concentration 20 times using its troponin kit, average is obtained, obtains flesh calcium egg respectively
The concentration of white calibration object：0. 2,0.67,1,3,4.8,10,27.3 ng/ml.
5th, the foundation of standard curve
6th, this kit is suitable for the various Immunofluorescence test instrument that can have excitation light source, and collect wavelength of transmitted light,
Design parameter can be adjusted according to instrument, substitute into calibration curve, you can calculate the content of troponin in sample to be tested.
2nd, the analytical performance assessment of detection kit
1, the range of linearity
With the troponin high concentration sample close to the range of linearity upper limit（20ng/ml), it is pressed 1/2 with physiological saline,
1/4,1/8,1/16,1/32,1/64 dilution is configured to the solution of 6 diluted concentrations, with the Biochemical Analyzer detection method altogether
Measure each diluted sample concentration.The average value of measurement result is obtained in each concentration replication 3 times respectively.Using diluted concentration as
Equation of linear regression is obtained using measurement result average value as dependent variable in independent variable.The related coefficient of linear regression is calculated by formula
R, the results show regression equation are y=0.9188X+0.5444, and correlation coefficient r=0.995 shows kit of the present invention 0.1
Good relationship in the ng/ml-30ng/ml ranges of linearity (see Fig. 3).
2nd, sensitivity (minimum detection limit）
The various concentration standard items calibrated according to standard items are added in clinical normal person's blood, measurement obtains standard curve, with
Normal mean value adds twice of standard deviation report minimum detection limit, and minimum detected value, the results show sheet is calculated by minimum detection limit
The lowest detection of invention kit is limited to 0. 1ng/ml.
Attached drawing 4 is and standard method detects blood sample contrasting data figure.
The anticoagulation new blood of below critical value 0.1ng/ml, according to 0,0.05,0.1,0.2,0.5,1,2,4,8,16
Concentration adds in Troponin I antigen（Purchased from Hytest companies）, respectively with Dimension RxL Max Integrated
Chemistry System （Siemens）Detection and present invention detection, obtain R=0.972, show that correlation is good.
Table 1 shows that detect blood sample obtains standard curve through the present invention, and calculates minimum detected value, repeats 3 samples,
Less than 0.1ng/ml, show that the present invention meets the requirement that the World Health Organization and professional institution formulate.
Table 1
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (5)
- A kind of 1. preparation method for quantitatively detecting Troponin I content immunofluorescent reagent box, which is characterized in that the quantitative inspection The preparation method for surveying Troponin I content immunofluorescent reagent box comprises the following steps：The first step, troponin antibodies-bulky grain fluorescent grain conjugate are prepared, and the use of phosphate buffer density are 10mM, are delayed Fliud flushing includes 0.39 gram of sodium dihydrogen phosphate, 1.02 grams of disodium hydrogen phosphate, 0.2 gram of azoles nitrogen sodium；It is 7.2 with salt acid for adjusting pH, adds BSA5 grams, make final concentration of：BSA5mg/ml, Proclin0.05%w/v；Second step, the preparation of nitrocellulose filter labelled antibody take CN140 nitrocellulose filter 30cm, are placed in point film instrument, It operates and requires according to point film instrument, antibody liquid is uniformly put on film with the speed of 1ml/cm；The nitrocellulose filter for being marked with antibody is placed in the position specified in suitable cartridge back box by the 3rd step, the assembling of reagent card It puts, is being sequentially placed into sample pad, then water absorption pad covers box in cartridge, be placed in sealing of the humidity not higher than 20% and close interior protect It deposits；4th step, TNI calibration objects are prepared, human troponin recombinant protein are dissolved in phosphate buffer and is added in 5% cow's serum, is made The calibration object of various concentration；Using troponin calibration object as primary standard, using troponin kit to the school of various concentration Quasi- product detect 20 times respectively, and average is obtained, and obtain the concentration of troponin calibration object：0.2,0.67,1,3,4.8,10, 27.3ng/ml；5th step, establishes standard curve；Above-mentioned 7 standard items are dissolved in into the phosphate buffer containing 5% cow's serum, then often Kind takes 75ul, instills in the sample well of test card, waits 10 minutes, has detector to read fluorescence signal, it is soft then to bring Excel into Part is calculated, and obtains standard curve；6th step is collected the data of wavelength of transmitted light by Immunofluorescence test instrument, substitutes into calibration curve y=0.9188x+ 0.5444, thus calculate the content of troponin in sample to be tested；The troponin antibodies-bulky grain fluorescent grain conjugate preparation specifically includes：Step 1, activation, the washing of latex particle, takes 2% aldehyde radical base latex latex particle solution, 100 μ 1, to latex solution Middle addition 0.6mg/mlNaBH4 solution I .1ml, are placed in 37 DEG C of reciprocal shakers and react 2h；12000rpm centrifuges 30min, abandons Supernatant adds in the washing of 50mMpH7.2 phosphate buffers three times, precipitation is scattered in 10mMpH7.2 phosphate buffers, makes latex Granule density is 1%w/v；Step 2, the crosslinking of large scale latex particle troponin antibodies, with 10mM phosphate buffers by 5mg/ml goat-anti people TNI polyclonal antibodies are diluted to the antibody-solutions of 1mg/ml, and adding in 400 μ l in the activation latex particle solution prepared in 1ml resists Liquid solution, puts 37 DEG C of shaking table lh, forms large scale latex particle-TNI polyclonal antibodies connection compound, 12500rpm speed from Heart 20min, abandons supernatant, and sediment is dissolved in O.1MpH7.4PBS buffer solution, mixing, and 12500rpm centrifugation 20min abandon supernatant, It then washes repeatedly twice；The 10mM phosphoric acid that final pellets are dissolved in the Proclin300 containing 4mg/mlBSA and 0.05% delays Fliud flushing；The preparation method of the latex particle comprises the following steps：Step 1, activation, the washing of bulky grain latex particle；The amino group of large particle surface modification is activated, by latex glue It is centrifuged after milk solution activation, abandons supernatant, precipitation is washed repeatedly 3 times with lavation buffer solution；Final pellets are resuspended in the washing In buffer solution, make the mass volume ratio final concentration of 1% of latex particle, the lavation buffer solution is selected from PBS buffer solution and MES delays One kind in fliud flushing；Step 2, Troponin I antibody coating latex particleThe latex solution after Iml activated rinses is taken, Troponin I antibody is added in, makes the final concentration of 1mg/ of Troponin I antibody ml；After mixing, 37 DEG C of shaking tables are put, 18h is coated with 220rpm rotating speeds；The Troponin I antibody not combined with latex particle is washed with PBS buffer solution, is repeated 3 times；Closing：The latex particle of coating Troponin I antibody after washing is dissolved in described comprising in protectant buffer solution, making The final concentration of 0.1-0.14% of mass volume ratio of latex particle；Closing after step 3, anti-human troponin antibodies and the combination of large scale latex particle, by large scale latex particle and institute Anti-human troponin antibodies mixture is stated, skimmed milk power 0.3% is added in, reacts 2h at room temperature, is centrifuged, removes wherein a small amount of sink Starch；Step 4, cleaning, by the large scale latex particle formed in step 3-anti-human troponin antibodies complex solution from The heart abandons supernatant, adds in phosphate buffer repeated washing 3 times；Final pellets are dissolved in slow containing the protective agent and preservative In fliud flushing, make the final concentration of 0.04%-0.14% of mass volume ratio of the latex particle.
- 2. prepared by a kind of preparation method for quantitatively detecting Troponin I content immunofluorescent reagent box as described in claim 1 Quantitatively detect Troponin I content immunofluorescent reagent box, which is characterized in that the quantitatively detection Troponin I content is immunized Fluorescence kit includes：Nitrocellulose filter, reagent and calibration object；The reagent includes surfactant, blocking agent, preservative and the latex particle for being coated with anti-human troponin antibodies, described It is connected between reagent moderate resistance human troponin antibody and large scale latex particle by peptide catenary system；The calibration object includes protection Agent, preservative, buffer solution and restructuring troponin；The mass ratio of the anti-human troponin antibodies and large scale latex particle is 1：10-1:100；The final concentration of 0.04-0.4% of latex latex particle mass volume ratio of the anti-human troponin antibodies of coating.
- 3. Troponin I content immunofluorescent reagent box is quantitatively detected as claimed in claim 2, which is characterized in that the examination The latex particle of a diameter of 390nm of bulky grain fluorescence carrier in agent；The one kind of the reagent moderate resistance human troponin antibody in polyclonal antibody and monoclonal antibody；For mouse source, rabbit source Or sheep source antibody；One or more of the protective agent in bovine serum albumin(BSA), ovalbumin and gelatin, the bovine serum albumin(BSA), Concentration is 10mg/ml；The troponin kit includes sample liquid, and sample liquid is selected from Tris-HCl buffer solutions, phosphate buffer and sweet One or more in propylhomoserin buffer solution；Required concentration is 10mM-100mM, pH 10；The troponin sample liquid uses phosphorus Acid buffer, concentration 10mM, pH 7.2.
- 4. Troponin I content immunofluorescent reagent box is quantitatively detected as claimed in claim 2, which is characterized in that the table Face activating agent is selected from polysorbas20, tween 100；The mass volume ratio of the surfactant is 0.02-0.4%；One or more of the preservative in Sodium azide, thimerosal and PrOClin300；The mass body of the preservative Product is than being 0.1-2%.
- 5. a kind of protect the preparation that Troponin I content immunofluorescent reagent box is quantitatively detected described in claim 1 any one The Immunofluorescence test instrument of quantitative detection Troponin I content immunofluorescent reagent box prepared by method.
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| CN109239361B (en) * | 2018-09-29 | 2022-02-08 | 迈克生物股份有限公司 | Detection kit for cardiac troponin I and preparation method thereof |
| CN111521825A (en) * | 2020-05-18 | 2020-08-11 | 巴迪泰（广西）生物科技有限公司 | Cardiac troponin I detection kit and detection method |
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| CN102841207A (en) * | 2011-06-24 | 2012-12-26 | 北京乐普医疗科技有限责任公司 | Fluorescent immune chromatographic test strip for quantitively detecting troponin I and preparation method thereof |
| CN102384973B (en) * | 2011-08-10 | 2014-09-10 | 华南理工大学 | Immunochromatography test strip for quickly and quantitatively detecting hydroxymethylfurfural and preparation method thereof |
| CN202631543U (en) * | 2012-06-26 | 2012-12-26 | 南京基蛋生物科技有限公司 | D-dimer fluorescent immune quantitative measurement test strip |
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| CN204165984U (en) * | 2014-10-28 | 2015-02-18 | 广州天宝颂原生物科技开发有限公司 | Cardiac muscle troponin I immunochromatographiassay assay quantitative detection test paper |
| CN104502595A (en) * | 2014-11-10 | 2015-04-08 | 江苏宏泰格尔生物医学工程有限公司 | Troponin detection method and kit thereof |
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